The B7-CD28/CTLA-4 costimulatory pathway has a critical role in regulating T cell activation, differentiation and tolerance, and is a promising therapeutic target. The recent discovery of the CD28 homologue ICOS, an inducible T cell costimulatory receptor, and its ligand, which is a B7 homologue, has revealed a new means by which T cell responses may be regulated, and raised questions about the functional significance of ICOS and its relationship with the B7-CD28/CTLA-4 pathway. Because ICOS is expressed only after TCR engagement, and ICOS costimulation induces IL-4 and IL- 10, which are important immunoregulatory cytokines, ICOS may have important influences on the amplification and/or regulation of an immune response. The goals of this project are to investigate the in vivo functions of ICOS and its functional relationship with the B7-CD28/CTLA-4 pathway: 1) We will examine the importance of ICOS for the generation of immune responses in vivo, by examining the functional consequences of ICOS dysregulation. We have generated anti-ICOS mAbs and ICOS-Ig and are generating mice lacking ICOS and transgenic mice that constitutively overexpress ICOS in T cells. These tools provide a means for determining when during an immune response ICOS exerts its effects. 2) We will use DO.11 TCR Tg T cells to visualize the impact of ICOS dysregulation on the activation, expansion, differentiation, and effector functions of antigen- specific CD4+ T cells. 3) We will analyze the inter- relationships between ICOS and the B7-CD28/CTLA-4 pathways during an immune response. We will examine whether these pathways reciprocally regulate the expression of each other, and evaluate functional relationships between the pathways. The availability of mouse strains lacking B7-1, B7-2 or both B7 costimulators, together with mouse strains lacking CD28, CTLA-4 or both receptors, provides us with unique opportunities to analyze the interactions between the ICOS-ICOS-CR and B7-CD28/CTLA-4 pathways. Taken together, these approaches should provide fundamental information regarding when during an immune response ICOS is needed and how ICOS interacts with the B7-CD28/CTLA-4 pathway. These studies may thereby assist in the design of optimal therapeutic strategies for manipulation of the B7- CD28/CTLA-4 pathway and also indicate the therapeutic potential of ICOS pathway manipulation.